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[Reprinted  from  the  Journal  of  the  American  Chemical  Society.  Vol.  XXVII. 
No.  3.  March,  1905.] 


ON  THE  PRESENCE  OF  COTTON-SEED  OIL  IN  LARDS 
FROiT  HOGS  FED  UPON  COTTON=SEED  iTEAL. 

By  A.  D.  Emmett  and  H.  S.  Grindley. 

Received  December  21,  1904. 

Having  had  occasion  to  make  for  the  Department  of  Animal 
Husbandry,  of  the  Experiment  Station  of  the  University  of 
Illinois,  quantitative  determinations  of  cotton-seed  oil  in  lards 
rendered  from  the  fat  of  hogs  fed  largely  upon  cotton-seed  meal, 
a more  detailed  study  of  the  samples  was  undertaken  in  order  to 
add,  if  possible,  some  information  as  to  whether  cotton-seed  oil 
actually  existed  in  the  body  fat  of  the  animals. 

That  many  of  the  vegetable  oils  enter  into  the  make-up  of  the 
body  fat  in  some  manner  or  other,  there  can  be  but  little  doubt. 
Lebedeff,1  and  Henriques  and  Hansen2  in  feeding  linseed  cake, 
Monk3  in  feeding  rape  oil  and  the  fatty  acids  from  mutton  tallow, 
Lebedeff4  and  Rosenfelt5  in  feeding  mutton  fat;  and  Shutt6  in 
feeding  maize,  have  shown  that  the  resulting  fat  differs  mate- 

1 Ztschr.  physiol.  Chem.  ,‘6,  149. 

2 Thier.  Chem.  Ber.,  29,  68. 

3 Ibid.,  14,  411. 

4 Ztschr.  Physiol.  Chem.,  6,  149. 

5 Thier.  Chem.  Ber.,  25,  44. 

6 Canada:  Cen.  Expt.  Sta.  Bull.,  38. 


^1.5by  1 


264 


A.  D.  EMMETT  AND  H.  S.  GRINDLEY. 


rially  from  the  normal  fat  body  in  appearance  and  properties — 
melting-point  and  iodine  number. 

On  the  other  hand,  Radziejewsky1  with  rape  seed  oil,  Subbotin2 
with  soap,  Tebedeff3  with  tributrine,  Henriques  and  Hansen4 
with  linseed  oil  on  cows,  Bomer,5  Harrington  and  Adriance,6 
Virchow,7  Van  Engelen,8  Solstein,9  Langfiirth,10  and  Fulmer11  with 
cotton-seed  meal,  were  unable  to  obtain  affirmative  results  in 
their  feeding  experiments,  although  in  almost  every  instance 
there  were  some  abnormal  changes  detected  in  the  fat.  In  the 
cases  of  cotton-seed  meal  some  of  the  investigators  found,  by  the 
Halphen  and  Bechi  colorimetric  tests,  as  high  as  15  to  30  per 
cent,  of  the  oil. 

Thus,  admitting  that  the  presence  of  vegetable  oils  influences 
the  fat  of  animals  to  some  extent,  the  question  arises  as  to  how 
these  oils  exist  in  the  body  fat.  Are  they  assimilated  unchanged 
through  the  lacteals  and  thus  find  their  way  into  the  circulatory 
system,  being  carried  on  to  the  fat  cells?  Are  they  first  broken 
up  into  simpler  bodies  and  then  absorbed  by  the  lacteals,  or  are 
they  subjected  to  the  intricate  processes  of  metabolism — saponi- 
fied by  the  bile,  etc. — and  converted  into  animal  fat? 

The  fact  that  Monk,  in  feeding  rape  oil  to  dogs,  not  only  found 
the  olein  content  of  the  body  fat  much  above  the  normal,  but 
that  he  was  able  to  separate  eruic  acid,  a characteristic  ingre- 
dient, tends  to  show  that  in  one  case,  at  least,  the  vegetable  oil 
was  evidently  absorbed  unchanged.  On  the  other  hand,  since 
Henriques  and  Hansen  found,  in  their  work  on  the  feeding  of 
linseed  oil  to  pigs  and  dogs,  that  the  same  ration  may  affect  the 
fat  of  one  animal  and  not  that  of  another;  and  since  Lebedeff 
in  feeding  the  same  animal  on  different  fats — linseed  oil  and 
tributrine — was  able  to  detect  a change  in  the  former  case  but 
none  in  the  latter,  it  tends  to  indicate  that  the  nature  of  the 
assimilation  of  the  rations  varies  under  certain  conditions — with 
the  animal  and  kind  of  animal  and  with  the  fat  and  kind  of  fat. 

1 Virchow’s  Archiv.,  56,  211;  43,  268. 

2 Ztschr.  f Biol.,  6,  73. 

3 Ztschr.  Physiol.  Chem.,  6,  149. 

4 Thier.  Chem.  Ber .,  39,  68. 

6 Ztschr.  Unt.  Nahr.  u.  Genus.,  1899,  2. 

6 Texas  Expt.  Sta.  Bull.,  29. 

7 Ztschr.  Unt.  Nahr.  u.  Genus.  (1899),  8,  46-48. 

8 Chem.  Centrbl.,  1899,  2. 

9 Zeit.  offentl.  Chem.,  8,  140. 

10  Ztschr.  angew.  Chem.,  14,  685. 

11  This  Journal,  24,  1148;  26,  837. 


COTTON-SEED  OIL  IN  LARDS. 


265 


To  answer  this  question  of  assimilation  in  the  case  of  cotton- 
seed meal-fed  hogs,  there  seemed  but  one  way  as  suggested  by 
Hehner1  in  1880,  when  European  markets  were  being  flooded  with 
adulterated  American  lards,  namely,  to  actually  establish  the 
presence  of  a vegetable  oil.  At  that  time,  the  solution  resolved 
itself,  in  the  case  of  cotton-seed  oil,  into  the  following  statement : 
If  a lard  gave  the  Bechi-Maumene  test  together  with  a high  iodine 
value  and  a low  melting-point,  it  should  be  condemned.  But  in 
cases  of  doubt,  Salkowski’s2  method  for  the  separation  of  vegetable 
cholesterin  should  be  applied.  Eater,  many  other  colorimetric 
tests  were  submitted,  chief  among  them  being  the  nitric  acid  and 
the  Halphen  tests.  Of  the  former,  Eewkowitsch3  stated,  previous 
to  the  appearance  of  the  latter,  that  he  preferred  it  to  all  others. 
Halphen’s  test,  however,  has  come  into  prominence  and  is  con- 
sidered to-day  to  be  the  most  convenient  and  reliable  method, 
both  from  the  qualitative  and  the  quantitative  standpoint,  for 
estimating  cotton-seed  oil,  and  by  many  it  has  been  given  the 
preference  to  the  once  official  Bechi  test. 

In  dealing  with  lards  from  hogs  fed  upon  cotton-seed  meal, 
the  factors  are  to  some  extent  different  from  those  in  the  case  of 
rendered  lards  mixed  with  cotton-seed  oil.  The  analytical  num- 
bers— iodine  absorption  value,  and  melting-point — are  not  trust- 
worthy, although  indicative.  Bomer4  has  shown  that  the  varia- 
tions in  the  iodine  number  make  this  test  impracticable.  Albert5 
and  likewise  Henriques  and  Hansen6  have  found  that  the  compo- 
sition of  the  fat  from  different  parts  of  the  same  animal  is  subject 
' to  great  differences  in  melting-point  and  iodine  number.  In  the 
case  of  Albert,  these  variations  were  greater,  on  the  average, 
than  could  be  ascribed  to  any  feeding  ration.  Hence,  in  this 
short  study,  we  have  directed  our  attention  chiefly  to  other  tests 
for  determining  qualitatively  the  presence  of  a vegetable  oil. 

One  of  the  supposed  essential  differences  between  animal  and 
vegetable  oils  is  in  their  monatomic  alcohols  called  cholesterins. 
The  former,  animal  cholesterin,  crystallizes  from  hot  alcohol  in 
rhombic  plates,  and  the  latter,  vegetable  cholesterin  and  desig- 

1 Analyst , 13,  165. 

2 Ztschr.  a?ial.  Chem.,  26,  557. 

3 “Oils,  Fats  and  Waxes,”  Benedickt  and  Lewkowitsch. 

4 Ztschr . Unter.  Nahr.  u.  Genus.,  1898,  532. 

5 Landw.  Jahrb.,  28,  961. 

6 Bied.  Centr.  Blatt.  Agr.  Chem.,  30,  182. 


266 


A.  D.  EMMETT  AND  H.  S.  GRINDLEY. 


nated  by  Hesse1  as  phytosterin,  crystallizes  in  needles  arranged 
in  stars,  fans  or  bundles.  According  to  Bomer,2  in  a mixture  of 
the  two  cholesterins,  the  phytosterin  either  takes  on  the  form 
of  elongated  plates  which  may  or  may  not  have  angles  at  the 
ends,  or  if  it  is  in  much  the  greater  proportion,  of  needles  which 
lie  upon  the  rhombic  plates  of  the  animal  cholesterin.  Van 
Kettel3  states  that  another  distinguishing  feature  of  some  veg- 
etable oils,  and  especially  cotton-seed  oil,  is  that  they  give  the 
pentosan  reaction.  In  this  connection  Rudzinski4  has  recently 
shown  that  the  pentosans  occur  in  company  with  the  carbohy- 
drates in  the  plant  cells  and  are  closely  connected  with  cellulose, 
a prominent  constituent  of  cotton-seed  meal.  Slovtzov5  also 
states  that  a part  at  least  of  pentosans  is  absorbed  by  the  animal 
body.  In  1891  Welman6  proposed  his  phosphomolybdic  test  for 
vegetable  oils,  suggesting  that  the  reaction  might  be  due  to  gluco- 
sides.  In  1901 7 he  defended  the  attacks  upon  his  test  and  added 
that  the  final  blue  coloration  which  would  also  result  if  amines 
were  present,  was  of  little  value,  as  the  characteristic  feature 
was  the  green  color  produced  when  the,  nitric  acid  was  added. 
Seiler  and  Verda8  have  lately  stated  that  Welman’s  reaction, 
though  not  a positive  test,  gives  good  indications  of  the  presence 
of  vegetable  oils  in  admixtures  of  animal  fats  or  mineral  oils. 

Again,  as  suggested  above,  Bomer  worked  upon  the  separation 
or  detection  of  a vegetable  oil  in  lards  from  cotton-seed  meal-fed 
hogs.  His  conclusions  were  that  inasmuch  as  he  could  not  obtain 
crystals  of  phytosterin,  the  vegetable  oil  was  not  retained  in  the 
animal  fat.  He  also  stated  that  since  a trace  of  the  fatty  acids' 
lowers  the  melting-point  of  the  crystals,  the  differences  in  their 
form  should  be  relied  upon  rather  than  that  of  the  melting-point. 
Virchow,  who  also  worked  upon  this  question,  obtained  results 
agreeing  with  Bomer  as  to  the  separation  of  phytosterin  and  the 
presence  of  a vegetable  oil  in  the  body  fat.  Solstein  in  his  work 
was  likewise  unsuccessful  in  isolating  the  vegetable  cholesterin  and 
also  in  obtaining  positive  results  with  the  Welman  reaction.  He 

1 Ann . Chem.  (niebig),  192,  175. 

2 Mitt.  d.  Kgl.  Techn.  Versuck.,  18,  255. 

8 Monat.  Scieniifique , 1900. 

4 Ztschr.  physiol.  Chem 40,  317. 

5 Ztschr.  Unter.  d.  Nahr.  u.  Genuss.,  1904,  p.  2. 

6 Pharm.  Ztg.,  36,  798. 

7 Ztschr.  f.  offentl.  Chem..  6,  143  ; 4,  852. 

8 Chem.  Ztg..  27. 


COTTON-SEED  OIL  IN  LARDS. 


267 


stated  that  a lard,  although  it  might  give  the  Halphen  test,  should 
not  be  condemned  unless  it  gave  Welman’s  reaction  and  crystals 
of  phytosterin. 

It  is  noteworthy,  at  this  point,  that  where  the  Halphen,  Bechi, 
and  nitric  acid  colorimetric  tests  hkve  been  applied  to  lards  from 
cotton-seed  meal-fed  hogs,  these  reactions  have  been  obtained 
almost  without  fail.  The  majority  of  investigators  agree  that 
each  of  these  characteristic  reactions  for  cotton-seed  oil  is  de- 
pendent upon  a specific  constituent  in  the  oil  for  its  ultimate 
color.  If  this  is  the  case,  then  the  animal  fat  has  apparently 
assimilated  these  three  bodies  in  their  unaltered  condition,  either 
through  the  medium  of  the  unchanged  cotton-seed  oil  or  its 
simpler  decomposition  products,  for,  should  they  have  been  sub- 
jected to  the  regular  processes  of  metabolism,  it  is  difficult  to  con- 
ceive that  they  could  have  been  transmitted  to  the  fat  cells 
without  one  of  them  having  lost  its  characteristic  property. 

In  our  work,  samples  of  leaf  and  ham  fat  were  obtained  from 
each  of  four  hogs  which  had  been  fed  largely  upon  cotton-seed 
meal.  The  fats  were  rendered  in  the  laboratory  at  a low  tempera- 
ture, not  exceeding  120°  C.  A sample  of  leaf  fat,  known  to  be 
free  from  cotton-seed  oil,  was  also  rendered  at  the  same  time 
and  under  the  same  conditions.  Quantitative  determinations  of 
cotton-seed  oil  were  then  made  on  each  of  the  eight  samples  of 
lard  by  the  Halphen  and  Bechi-Hehner  tests.  The  amount  of 
cotton-seed  oil  was  found  to  range  from  5 to  15  per  cent.  The 
Bechi-Hehner  method  gave  somewhat  higher  results  throughout. 

The  first  four  samples  of  the  above  lards  were  then  taken  for 
the  more  detailed  study  as  to  the  presence  of  the  vegetable  oil. 

In  applying  the  nitric  acid  tests  to  these  lards,  the  character- 
istic brown  color  appeared  at  once  and  was  very  pronounced.  A 
blank  test  upon  the  pure  lard  gave  no  color,  even  after  standing 
twenty-four  hours. 

As  suggested  by  Van  Kettel,  Tollen’s1  phloroglucinol  hydro- 
chloride reaction  for  pentosans  was  tried.  With  each  of  the  sam- 
ples a distinct  reddish  pink  ring  was  noticeable  at  the  juncture 
of  the  two  layers.  A parallel  determination  upon  the  pure  sample 
gave  no  such  results,  showing  that  pentosans  were  present  in  the 
lards  in  question. 

Welman’s  phosphomolybdic  test  was  carefully  tried.  The 

1 Jour.f  Landw.,  40,  13. 


268 


A.  D.  EMMETT  AND  H.  S.  GRINDLEY. 


characteristic  green  and  blue  colorations  were  obtained  repeatedly 
when  applied  to  the  samples  in  question,  but  in  no  case  could  they 
be  produced  with  the  pure  lard. 

Having  been  successful  in  these  characteristic  tests,  attention 
was  then  directed  to  the  separation  of  the  vegetable  cholesterin, 
phytosterin,  believing,  with  Salkowski,  Hehner,  Bonier,  Solstien 
and  others,  that  should  this  unsaponifiable  substance  be  found, 
the  evidence  would  be  still  stronger  in  favor  of  the  presence  of  a 
vegetable  oil  in  the  lards. 

Three  attempts  were  made  to  separate  the  phytosterin.  First, 
Von  Raumer’s1  method  was  found  to  yield  a substance  which 
agreed  fairly  well  with  the  description  of  phytosterin,  but  the 
quantity  obtained  was  so  small  that  no  positive  results  could  be 
procured.  Forster  and  Riechelman’s2  method  was  then  applied. 
The  yield  was  somewhat  larger  than  in  the  previous  case  and  the 
same  characteristic  features  concerning  the  crystalline  formation 
were  noticeable.  In  this  instance,  a parallel  determination  was 
made  upon  a sample  of  cotton-seed  oil.  There  was  a similarity 
in  their  crystalline  structure,  but  we  were  still  dissatisfied  with 
the  results.  In  a somewhat  recent  article  by  Ritter,3  he  has  shown 
that  his  method  for  the  separation  of  cholesterin  from  fats  will 
give  a larger  yield  than  any  of  the  others.  We  were  led  to  try 
this  procedure  in  our  case.  Briefly  stated,  it  is  as  follows : Fifty 
grams  of  the  fat  are  heated  in  a porcelain  dish  upon  a water- 
bath  with  ioo  cc.  of  alcohol.  This  solution  is  saponified  by  the 
addition  of  a hot  mixture  of  8 grams  of  sodium  hydroxide 
and  160  cc.  of  strong  alcohol.  It  is  then  evaporated  and 
75  grams  of  sodium  chloride  are  added  and  the  whole  is 
dissolved  in  hot  water.  The  solution  is  brought  to  dryness  with 
continued  stirring  and  the  residue  pulverized,  and  left  over  sul- 
phuric acid  for  some  time.  The  dried  mass  is  then  extracted 
with  ether  for  at  least  nine  hours,  using  a Soxhlet  apparatus. 
The  extract  is  freed  from  ether  at  a low  temperature  and  the 
residue  of  cholesterin  is  finally  taken  up  with  hot  alcohol  and 
allowed  to  cool  slowly,  when  crystals  will  separate. 

Two  hundred  grams  of  the  lard  in  question  and  50  grams  of 
pure  cotton-seed  oil  were  carefully  treated  as  just  stated.  As  a 
result,  we  obtained  a much  larger  yield  than  in  either  of  the  other 

1 J,  Soc.  Chem.  Ind 1898,  p.  774. 

2 Analyst , 1897,  p.  131. 

3 Ztschr.  physiol.  Chem.,  34,  461. 


COTTON-SEED  OIL  IN  LARDS. 


269 


two  methods,  although  the  amount  obtained  in  this  case  was 
still  small.  The  crystals  from  the  evaporation  of  the  ether  were 
needles.  After  treatment  with  hot  alcohol  and  allowing  the  crys- 
tallization to  continue  slowly,  needles  again  separated.  In  the  case 
of  the  cotton-seed  oil,  they  were  arranged  in  stars,  and  in  the  case 
of  the  lard,  in  leaf-like  forms.  Upon  recrystallization,  the  stars 
retained  their  same  general  shape,  but  the  leaf-like  forms  changed 
into  bundles  of  needles.  These  bundles  crossed  each  other  at 
different  angles,  forming  clusters.  A second  recrystallization  of 
these  bundles  gave  crystals  of  a distinct  fan  shape  with  a star 
of  needles  as  a base  and  having  the  fascicular  portion  built  up 
from  this  nucleus.  By  recrystallizing  these  again,  they  passed  into 
the  previous  form — bundles  of  needles.  Salkowski’s  chloroform- 
sulphuric  acid  test  for  cholesterin  was  applied  to  this  small  quan- 
tity of  crystals.  The  test  proved  the  presence  of  a cholesterin 
and  the  crystalline  form  indicated  that  it  was  probably  phytosterin. 

However,  as  a further  confirmation  of  the  presence  of  phyto- 
sterin, Bomer  ascertains  the  melting-point  of  the  acetic  esters  of 
these  crystals.  He  states1  that  this  method  is  a very  sensitive  one 
for  detecting  the  kind  of  cholesterin.  If  the  melting-point  of  the 
crystals  is  1160  C.  or  higher,  it  shows  phytosterin  to  be  present. 
This  method  has  been  used  by  Tolman2  in  attempting  to  identify 
phytosterin  in  lard  from  cotton-seed  fed  hogs,  in  which  he  ob- 
tained negative  results.  Juckenack  and  Pasternack3  agree  with 
Bomer  in  regard  to  the  acetate  test  for  phytosterol,  but 
they  do  not  make  any  statement  as  to  its  delicacy.  On  the 
other  hand,  Siegfeld4  states  that  while  the  test  is  accurate,  it  is 
not  sensitive  in  the  cases  of  the  fats  with  which  they  worked — 
margarin  and  palmitin — to  less  than  10  per  cent.  Bomer  claims 
that  cotton-seed  and  sesame  oils,  which  are  frequently  present 
to  considerable  extent  in  margarin,  can  be  detected  by  this  test 
when  present  to  the  extent  of  only  1 to  2 per  cent.  It  is  thus 
apparent  that  there  is  still  a difference  of  opinion  as  to  the  delicacy 
of  this  test.  In  our  work,  as  a result  of  repeated  experiments,  we 
always  obtained  crystals  which  closely  resembled  those  of  phyto- 
sterin but  as  yet  we  have  not  been  able  to  prove  conclusively  the 
presence  of  this  substance  by  the  melting-point  of  its  acetate. 

1 Ztschr.  Unter.  Nahr.  u.  Genus.,  4,  1901;  5,  1902. 

2 Science , 1904,  763. 

3 Ztschr.  Unter.  Nahr.  u.  Genus.,  7,  1904. 

4 Ibid..,  7,  1904. 


270 


COTTON-SEED  OIL  IN  LARDS. 


This  brief  study,  which  was  originally  undertaken  in  order  to  con- 
firm the  Halphen  and  Bechi  tests  for  cotton-seed  oil  in  lards  from 
hogs  fed  upon  cotton-seed  meal,  has  been  found  to  present  so 
many  subjects  of  interest  upon  which  as  yet  very  little  has  been 
published  that  we  are  now  making  still  further  investigations  upon 
the  problems  involved. 

conclusions. 

These  several  samples  of  lard  rendered  from  the  fat  of  cotton- 
seed meal-fed  hogs,  were  found  to  give  the  following  tests  for 
vegetable  oils:  (I)  Welman’s  reaction;  (II)  Tollen’s  pentosan  re- 
action; (III)  Salkowski’s  cholesterin  reaction ; and  (IV)  crystals  re- 
sembling phytosterin.  The  samples  also  gave  the  following  tests  for 
cotton-seed  oil : (I)  the  Bechi ; (II)  the  nitric  acid ; and  (III)  the 

Halphen  reactions.  From  this  it  is  evident  that  two  statements 
can  be  made,  first  that  the  lards  contain  a vegetable  oil,  and 
second,  if  we  agree  with  the  most  recent  authorities,  they  contain 
three  distinct  constituents  of  cotton-seed  oil.  Hence,  it  seems 
safe  to  say  that  a part  at  least,  of  the  oil  existing  in  cotton-seed 
meal,  is  absorbed,  in  the  case  of  hogs  fed  upon  this  ration,  by 
the  animal  body  and  transmitted  in  its  unaltered  condition 
to  the  fat  cells. 


